Bombesin-induced poikilothermy in rats

Brain Research, 188 (1980) 525-530 © Elsevier/North-Holland Biomedical Press

525

BOMBESIN-INDUCED P O I K I L O T H E R M Y IN RATS

YVETTE TACHt~*, QUENTIN P1TTMAN* and MARVIN BROWN The Peptide Biology Laboratory and ( Q.P.) Arthur Vining Davis Center for Behavioral Neurobiology, The Salk Institute, P.O. Box 85800, San Diego, Calif. 92138 (U.S.A.)

(Accepted September 27th, 1979) Key words: bombesin - - peptide - - thermoregulation

SUMMARY The effect of ambient temperature (TA) on the rectal temperature (TRE) response to intraventricular injection of bombesin has been evaluated in conscious adult male rats. At TA = 4 °C, bombesin (50 ng-1 #g) caused a marked hypothermia which was dose-dependent both in terms of the magnitude and of the duration of the response. The bombesin-induced hypothermia was reduced at T.~ = 24 °C, whereas at TA ---- 31 or 33 °C, the peptide (1 #g) failed to affect TRE. At TA = 36 °C, bombesin 1-10/zg induced an elevation in TRy. The hyperthermia observed at high TA could be reversed to hypothermia by transferring rats to cold. The analogs [D-Trpa]bombesin or [D-Leu13] bombesin, tested under the same conditions, failed to produce significant changes in TRy. These findings demonstrate that bombesin appears to act in the brain as a poikilothermic agent by disrupting thermoregulation at temperatures below or above thermoneutrality.

INTRODUCTION Bombesin, a naturally occurring tetradecapeptide originally isolated from anuran skin 1, exerts potent effects on the central nervous system (CNS). Bombesin given intracranially has marked effects on thermoregulation2-4,s, is, glucoregulation 5,9,2°, adenohypophyseal hormone secretionta, 19, sympathetic activity7, 9 and gastric function18, 20. With the use ofimmunofluorescence and radioimmunoassay methods, the presence and distribution of bombesin-like activity has been described in mammalian brain, gastrointestinal tract and lung 6. Recently, high affinity binding sites for bombesin have been demonstrated in rat brain synaptosomal preparations 14. These various observations * Recipients of the Canadian Medical Research Council Centennial Fellowship.

526 raise the possibility of an endogenous bombesin-like ligand, present in the brain and exerting neurotropic actions. Brown et al. have demonstrated that bombesin is one of the most potent substances known to affect thermoregulation 2. The peptide produces a marked hypothermia when injected intracisternally under ether anesthesia in cold-exposed rats2-4,s, 18, whereas at room temperature, bombesin exerts no 2 or a diminished hypothermic effect5. The investigation reported here was undertaken to further clarify bombesin's action on thermoregulation by evaluating the influence of ambient temperatures on the rectal temperature response following injection of bombesin in conscious rats. MATERIALS AND METHODS Animals Adult, male Sprague-Dawley rats weighing 300-350 g were maintained ad libitum on Purina laboratory chow and tap water, and were housed under conditions of controlled temperature (22-23 °C) and illumination (from 06.00 to 22.00 h). All the experiments were begun between 11.00 and 13.00 h. A chronic cannula was placed in the right lateral brain ventricle under pentobarbital anesthesia (50 mg/kg) according to the method of Hayden et al. lz. Following a minimum of 5 days recovery, rats were placed in individual cages in an environmental chamber where ambient temperature (Ta) could be precisely regulated ( ± 1 °C). Unless otherwise indicated, all animals were exposed to the indicated ambient temperatures for one hour before peptide injection. Rectal temperature (TRg) was recorded with a Yellow Springs International Thermistor probe inserted 5 cm into the rectum and held in place until a stable reading was obtained. TR~ was taken 1 h before peptide injection and at 30 min intervals thereafter for up to 4 h. In some experiments, skin temperature was also monitored with a skin thermistor (YSI) taped to the base of the rat's tail. Peptides synthesized using solid phase methodology 17 were dissolved in sterile, pyrogen-free artificial CSF 2 and administered intracerebroventricularly (ivt) in 10 #1 volume to conscious rats. The animals were unrestrained during all the experiments except for the brief periods required for peptide injection and TRE measurements. Any obvious changes in behavior (e.g. eating, drinking, excessive locomotor activity) after drug injections were noted. Data were subjected to analysis of variance and differences between control and bombesin treatments were evaluated by the multiple range tests of Dunnett and Duncan using the computer program EXBIOL. RESULTS Body temperature responses to ivt injection of bombesin and the analogs [TrpS]and [Leula]-bombesin were examined in rats kept at TAS of 4, 24, 31, 33 and 36 °C. At no time did animals receiving the bombesin analogs show a significant change in TgE (Figs. 14).

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Fig. 1. Effect on rectal temperature of bombesin (1 or 0.05/~g) and bombesin analog [D-Leuta]-bombesin (1/~g) injected ivt at time = 0, to conscious rats at an ambient temperature of 4 °C. Each point represents the mean 4- S.E.M. (vertical bars). Values significantly different from 0 min values: *P < 0.05; **P < 0.01.

The time course of the hypothermic response to bombesin injected ivt into conscious rats exposed at 4 °C is shown in Fig. 1. It can be seen that both the extent and duration of the hypothermia are dose-dependent. After the injection of 0.05/ag bombesin, the core temperature of rats decreased maximally by 2 °C and returned to normal in 2 h. The 1 y g dose produced a deeper hypothermia which was maximal at 2 h ( - - 5 °C); TR~ returned to baseline levels 4 h post-injection. 39 Saline (N=I2)

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Fig. 3. Effect of different ambient temperatures on the rectal temperature of conscious rats measured I h after injection of 1/~g bombesin or bombesinanalog [o-TrpS]-bombesin.Each point represents mean rectal temperature of 5-11 animals, **P < 0.01 as compared with bombesin analog-treated rats. In rats kept at 24 °C, bombesin (1/~g) also elicited a significant fall in rectal temperature (Fig. 2). However, the maximum temperature response ( - - 2 °C) and the duration were considerably less compared with the same dose given to rats exposed at 4 °C. The decrease in TRE was accompanied by an elevation in tail temperature (Fig. 2). As shown in Fig. 3, bombesin or bombesin analogs (1 #g dose) did not modify TR• when injected into ratskept in a warm room (31 or 33 °C) throughout the experimental period. However, in rats exposed to 36 °C bombesin induced a significant rise in body temperature ( + 1.2 °C) for the duration of the experiment. Higher doses of bombesin (10 #g) injected into rats kept at 36 °C did not further enhance the hyperthermic effect (data not shown). Fig. 4 illustrates that bombesin injection (1 Fg ivt), followed by 1 h exposure to 36 °C, induced an increase in body temperature ( + 1.2 °C) which was counteracted by transferring rats to cold (4 °C). The maximal drop in body temperature under these conditions was --3.3 °C.

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529 At all TAs tested bombesin administration was followed by a transient stereotypic scratching as previously described2, but no other obvious behavioral effects (e.g. drinking, feeding, panting, saliva spreading, huddling) were noted. DISCUSSION This study indicates that the nature and magnitude oftbe effect of the bombesin on TRS is influenced by Ti. Confirming and extending previous findings~-4,s, is, we have demonstrated a reversible hypothermic effect of bombesin administered ivt to unanesthetized rats kept at 4 °C. At TA within the classic definition of the rats' thermoneutral zone (28-32 °C15), bombesin administration was without effect on TRE, whereas in rats exposed at high TA, a hyperthermic action for this peptide was revealed (Fig. 3). These findings demonstrate that bombesin appears to act as a poikilothermic agent by disrupting thermoregulation at temperatures above or below thermoneutrality. Consistent with this finding is the observation that the hyperthermia that developed in bombesin-treated rats exposed at 36 °C for 1 h could be reversed to a hypothermic response when rats were transferred to a cold environment. Bombesin analogs [D-Leula]- and [D-TrpS]-bombesin, previously shown to be devoid of hypothermic effect in cold-exposed rats TM, and of binding affinity for specific bombesin receptors in rat brain membrane 14, failed to induce significant changes in core temperature in rats exposed at low or warm environments. These results confirm the specificity of bombesin's action on thermoregulation. Accumulating experimental evidence seems to indicate that bombesin acts within the central nervous system2-3,10,11. However, the mechanism by which bombesin modifies body temperature remains to be established. In goats, bombesin is a potent thermolytic agent, evoking physiological and behavioral heat loss,and suppressing metabolic heat gain through inhibition of pituitary-thyroid activation and shivering10,n. In rats, bombesin completely abolished the plasma TSH rise induced by cold exposure4, s and we now report a reduction in heat conservation through tail vasodilation. Further studies are required to determine if these effects are due to bombesin's action on the central effector mechanisms involved in maintaining thermal balance or whether bombesin alters thermal sensation. The present study showing both hypo- and hyperthermic responses to bombesin would suggest that bombesin does not act in the rat to change the preferred temperature about which body temperature is regulated. ACKNOWLEDGEMENTS This work was supported by the NIH (NINCDS) Research Grant 14263-01. The skillful laboratory assistance of Miss G. Berg and Mr. R. Kaiser and the secretarial help of Miss S. Hebert and Mr. J. Zwicker are gratefully acknowledged.

530 REFERENCES 1 Anastasi, A., Erspamer, V. and Bucci, Isolation and structure of bombesin and alytesis, two analogous active peptides from the skin of the European amphibians Bombina and Alytes, Experientia (Basel), 27 (1971) 166-167. 2 Brown, M., Rivier, J. and Vale, W., Bombesin: potent effects on thermoregulation in the rat, Science, 196 (1977) 998-1000. 3 Brown, M., Rivier, J. and Vale, W., Actions of bombesin, thyrotropin releasing factor, prostaglandin Ea and naloxone on thermoregulation in the rat, Life Sci., 20 (1977) 1681-1688. 4 Brown, M., Rivier, J. E., Wolfe, A. I. and Vale, W. W., TRF and bombesin: actions on thermoregulation and TSH secretion in rats, Endocrinology, 100 (1977) 265 (abstract). 5 Brown, M.,Rivier, J. andVale, W.,Bombesinaffectsthe central nervous system to produce hyperglycemia in rats, Life ScL, 21 (1978) 1729-1734. 6 Brown, M., Allen, R., Villarreal, J., Rivier, J. and Vale, W., Bombesin-like activity: radioimmunologic assessment in biological tissues, Life Sci., 23 (1978) 2721-2728. 7 Brown, M. and Fisher, D., Plasma catecholamines- regulation by brain peptidergic and cholinergic systems, Endocr. Soc., 61 (1979) 125. 8 Brown, M. and Vale, W., Peptide effects on thermoregulation. In Pharmacology of Thermoregulation, 4th Int. Symp., Oxford, in press. 9 Brown, M., Tach6, T. and Fisher, D., Central nervous system action of bombesin: mechanism to induce hyperglycemia, Endocrinology, in press. 10 Gale, C. C., Singer, A., Sekijima, J., Yamiuchi, P., McGreery, B. and Hampson, N., Bombesin inhibits the cold-induced pituitary-thyroidresponse in goats, Endocr. Soc., 61 (1979) 187 (abstract 459). 11 Gale, C. C. and McCreery, B. R., Mechanisms of bombesin hypothermia, Fed. Proc., 38 (1979) 977 (abstract 3938). 12 Hayden, J. F., Johnson, L. R. and Maickel, R. P., Construction and implantation of a permanent cannula for making injections into the lateral ventricle of the rat brain, Life Sci., 5 (1966) 1509-1515. 13 Miyata, M., Guzman, S., Rayford, P. L. and Thompson, J. C., Effect of bombesin on release of gastrin, CCK, secretion and pancreatic exocrine secretion, Gastroenterology, 74 (1978) 1137A. 14 Moody, T. W., Pert, C., Rivier, J. and Brown, M., Bombesin: specific binding to rat brain membranes, Proc. nat. Acad. Sci. (Wash.), 75 (1978) 5372-5376. 15 Poole, S. and Stephenson, J. D., Body temperature regulation and thermoneutrality, Quart. J. exp. Physiol., 62 (1977) 143-149. 16 Rivier, C., Rivier, J. and Vale, W., The effects of bombesin and related peptides on prolactin and growth hormone secretion in the rat, Endocrinology, 102 (1978) 519-522. 17 Rivier, J., Somatostatin: total solid phase synthesis, J. Amer. chem. Soc., 96 (1974) 2986-2992. 18 Rivier, J. and Brown, M., Bombesin, bombesin analogs and related peptides: effects on thermoregulation, Biol. Chem., 17 (1978) 1766-1771. 19 Tach6, Y., Brown, M. and Rollu, R., Effects of neuropeptides on adenohypophyseal hormone response to acute stress in rats, Endocrinology, 105 (1979) 220-224. 20 Tach6, ¥., Simard, P. and Collu, R., Prevention by bombesin of cold-restraint stress induced hemorrhagic lesions in rats, Life Sci., 24 (1979) 1719-1726.